The slightly modified method described by Tofighi et al.146 was used to determine the total cardiac glycoside content of the C. gigantea stem bark extracts. Briefly, each extract (1 mg) was dissolved in 50% aqueous ethanol (1 mL) and then mixed with Baljet's reagent (1 mL), which was freshly prepared [1% picric acid (95 mL) mixed with 10% NaOH solution (5 mL)]. The mixture was allowed to stand in the dark at room temperature (30 ± 5 °C) for 1 h before being diluted with purified water (2 mL). The absorbance (482 nm) of the reaction solution was measured by using a UV/Vis spectrophotometer (Shimadzu UV-1800, Japan). The total cardiac glycoside content of each extract was calculated from the calibration curve of digoxin (Sigma–Aldrich, USA, 5–50 µg/mL, Y = 0.018X + 0.03, R2 = 0.9945, where Y represents the absorbance of digoxin at 482 nm, X represents the digoxin concentration (µg/mL), and R2 is the linear correlation coefficient. The average values ± standard deviation values (S.D.) from three independent experiments are reported in milligrams of digoxin equivalents per gram of extract (mg DXE/g extract).
Digoxin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, France
Digoxin is a laboratory product used for the detection and measurement of digoxin levels in biological samples. It is a cardiac glycoside extracted from the foxglove plant (Digitalis purpurea) and is commonly used in the management of certain heart conditions. The product provides a standardized and reliable method for quantifying digoxin concentrations, which is essential for therapeutic drug monitoring and patient care.
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114 protocols using digoxin
1
Quantification of Cardiac Glycosides in C. gigantea
2
Cardiac Glycoside Quantification in Extracts
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The method described by Tofighi et al. [21 ] was slightly modified and used to determine the total cardiac glycoside content of the samples. One milliliter of sample solution in 50% ethanol (1 mg/mL) was added to one mL of freshly prepared Baljet's reagent [1% picric acid (95 mL) mixed with 10% NaOH solution (5 mL)]. The reaction mixture was incubated at room temperature (30 ± 5 °C) in the dark for 1 h, diluted with 2 mL of purified water, and then the absorbance was measured at 495 nm using a UV/Vis spectrophotometer (Shimadzu UV-1800, Japan). The total cardiac glycoside content was calculated from a calibration curve of digoxin (Sigma‒Aldrich, USA, 5–50 μg/mL, as following Eq. (1): Y = 0.0153X+0.0502, R2 = 0.9990, where Y, X and R2 represent the absorbance of digoxin at 495 nm, the concentration of digoxin (μg/mL) and the linear correlation coefficient, respectively). The total cardiac glycoside content was presented as milligrams of digoxin equivalents per gram of extract (mg DXE/g extract). The independent experiments were performed three times.
3
Quantifying Cardiac Glycosides in Extracts
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The total cardiac glycoside content was determined using a modification of the method described by Tofighi et al. [26 ]. The sample solution (1 mL, 1 mg/mL in ethanol: water 1:1) was mixed with 1 mL of freshly prepared Baljet’s reagent (95 mL of 1% picric acid mixed with 5 mL of 10% NaOH solution). The reaction mixture was left in the dark at room temperature (30 ± 5°C) for 60 minutes. It was then diluted with purified water (2 mL), and the absorbance was measured at 495 nm using a UV/Vis spectrophotometer (Shimadzu UV-1800, Japan). The total cardiac glycoside content was calculated using a digoxin (Sigma-Aldrich, USA) calibration curve in the range of 5–50 μg/mL (Y = 0.0154X + 0.05, R2 = 0.9989, where Y represents the absorbance of digoxin at 495 nm, X represents the concentration of digoxin (μg/mL), and R2 represents the linear correlation coefficient). The cardiac glycoside content was expressed as milligram digoxin equivalents per gram of extract (mg DXE/g extract). The experiments were performed in triplicate.
4
Hypoxia and Digoxin Effects on Neuroblastoma Cells
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The human NB lines SK-N-AS and SK-N-BE(2)C (ECACC Nos 94092302 and 95011817) were grown in minimal essential medium supplemented with 10% foetal calf serum and 1% non-essential amino acids (both Life Technologies, Carlsbad, CA, USA) and maintained in a humidified incubator at 37 °C with 5% CO2. For hypoxic studies, cells were maintained at 37 °C with 5% CO2 and 1% O2 (Don Whitley Scientific, Shipley, UK; Hypoxystation-H35) or 8% (Eppendorf, Hamburg, Germany; Galaxy 48 R). For DMOG treatment, cells were cultured in media supplemented with 0.5 mm DMOG (Enzo Laboratories, Farmingdale, NY, USA) for 24 h. For digoxin treatment, 5, 10 or 100 nm digoxin (Sigma-Aldrich, St Louis, MO, USA) were added to the cells on day 0 followed by incubation as indicated.
5
iPSC-CM Contractility Imaging and Stress Analysis
6
Evaluating Digoxin's Impact on Cancer Stem Cells
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Preliminary experiments were performed to determine the optimal Digoxin concentrations to use in both CSC-enriched populations. For the analysis in spheres, the cells were treated for 1 h at 37°C with Digoxin (Sigma-Aldrich) at 5 nM. TZ (8 µM) was then added for 48 h at 37 °C. For the analysis after sphere dissociation, the cells were treated for 1 h at 37 °C with Digoxin (Sigma-Aldrich) at 5 nM for CSC-UMR and 2.5 nM for CSC-MN. TZ (30 µM) was then added for 3 h at 37 °C. Live/Dead® Viability/cytotoxicity Kit for mammalian cells (Molecular probes, Thermofisher Scientific, France) was used to determine the percentage of cell death in the absence and in the presence of Digoxin. Cells were incubated for 30 min at room temperature with Live/Dead solution (PBS, 0.5 µM Calcein-AM, 1 µM Ethidium homodimer-1). Cells were observed using a fluorescent microscope (Axiovert, Zeiss). Between 500 to 600 cells were counted for each condition (seven random fields for each slide) in two independent experiments.
7
Conditional Knockout of Wwox in Hepatocytes
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Wwox-floxed (Wwoxfl/fl) C57BL6/J;129sv mixed genetic background mice were bred with Albumin-Cre transgenic mice to generate Wwox conditional knockout in hepatocytes (WwoxΔHep mice). Male pups of control and WwoxΔHep mice were IP injected with 5 mg/Kg DEN (Sigma Aldrich) at the age of 14 days. Partial hepatectomy (30%) of the liver was done on 4-month-aged males as described72 (link). For digoxin treatment, digoxin (1 mg/Kg, Sigma Aldrich) was IP injected into DEN-treated mice starting at age of 6.5 months, 3-times/week, for a total of 8-months. The mice in HFD experiment were fed by 60% kcal fat (Research Diets INC, D12492) for 30 weeks. Tumor load was assessed by liver weight in grams. All experiments involving mice were approved by the Hebrew University Institutional Animal Care and Use Committee.
8
Fluorescent Probe Assay for Transporter Inhibition
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5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDCF) and 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDCFDA) were purchased from AAT Bioquest (Sunnyvale, CA, United States). Digoxin, estrone 3-sulfate (E3S), and lopinavir were purchased from Sigma-Aldrich (St. Louis, MO, United States). Nelfinavir was obtained from APExBIO Technology (Houston, TX, United States). Darunavir was purchased from Toronto Research Chemicals (North York, ON, Canada). A rabbit monoclonal P-gp antibody (ab120904), a rabbit monoclonal MRP2 antibody (ab172630) and a rabbit monoclonal GAPDH antibody (ab181602) were purchased from Abcam (Cambridge, MA, United States), and a rabbit monoclonal BCRP antibody (CST42078) was obtained from Cell Signaling Technology (Danvers, MA, United States). All other chemicals and solvents were of the highest grade commercially available.
9
Comprehensive Pharmacological Compound Database
10
Peroxynitrite Synthesis and Antioxidant Assays
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Chloroform, methanol (isocratic grade) and acetonitrile (LC–MS grade) were purchased from Merck (Darmstadt, Germany). Formic acid (LC–MS grade) and digoxin (internal standard, IS) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ultrapure water was prepared using a Milli–Q water purification system (Millipore, Milford, CT, USA). Peroxynitrite was synthesized according to the method of Pryor et al. [40 (link)]. DPPH, Trolox®, rosmarinic acid, thiobarbituric acid, Histopaque®–1077 medium, indomethacin, penicillin–streptomycin solution for cell culture and resazurin (In vitro toxicology assay kit, resazurin based, TOX8–1KT) were also purchased from Sigma–Aldrich (St. Louis, MO, USA). COX (human) Inhibitor Screening Assay Kit (Item No. 701230) and COX Colorimetric Inhibitor Screening Assay Kit (Item No. 701050) were from Cayman Chemicals (Ann Arbor, MI, USA). Reagents and cuvettes for cytotoxicity assays were purchased from NanoEnTek Inc. (Seoul, Korea). All other reagents were provided by local or international suppliers (mainly Avantor Performance Materials, Gliwice, Poland).
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